Humans with the polyendocrine autoimmune disease APECED (or APS-1) harbor mutations in the AIRE gene, which encodes a protein with the structural and functional features of a transcriptional regulatory factor. Mice carrying an engineered null mutation of the a/re locus also develop multi- organ autoimmune disease. Mechanistic studies performed on these mice during the last funding cycle of R01 DK60027 established that aire protects an organism from autoimmunity by promoting the negative selection of differentiating thymocytes. It operates primarily by inducing the expression of transcripts encoding peripheral-tissue antigens, or PTTs, specifically in thymic medullary epithelial cells (MECs). Secondly, it enhances the capacity of MECs to present antigens to T cells and to clonally delete self-reactive thymocytes - via a currently unknown mechanism dissociable from PTT induction. This competitive renewal application proposes to further elucidate these two aspects of aire's mode of action - specifically, we intend: i) to gauge the plasticity of the MEC PTT repertoire; ii) to generalize and localize the antigen-presentation defect exhibited by aire-null MECs; iii) to assess the relevance of aire's role as an E3 ubiquitin ligase. Results from these studies should elucidate critical mechanistic elements of centrally (i.e. thymically) mediated immunological tolerance, potentially improving prospects for preventive or curative therapy for autoimmune diseases such as type-1 diabetes and multiple sclerosis.